Báo cáo Y học: Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes

Eur. J. Biochem. 269, 3751–3759 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03074.x Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes Ulrike Krause*, Luc Bertrand and Louis Hue Hormone and Metabolic Research Unit, Christian de Duve International Institute of Cellular and Molecular Pathology and University of Louvain Medical School, Brussels, Belgium Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 riboso-mal protein S6 kinase (p70S6K)and acetyl-CoA car-boxylase (ACC)involved in protein and fatty acids synthesis, respectively. In contrast, the AMP-activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosyn-thetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino-acid-induced acti-vation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by incubation with 5-aminoimidazole-4-carboxyamide ribo-nucleoside (AICAr)or oligomycin, an inhibitor of mito-chondrial oxidative phosphorylation. Incubation of hepatocytes with amino acids activated p70S6K via multiple phosphorylation. It also activated ACC by a phosphatase-dependent mechanism but did not modify AMPK activation. Conversely, the amino-acid-induced p70 ribosomal protein S6 kinase (p70S6K)participates in the control of protein synthesis and is activated in response to hormones, mitogens and nutrients (reviewed in [1–3]). It phosphorylates the 40S ribosomal protein S6, which is involved in the translation of certain mRNAs, the so-called 5¢-TOP mRNAs encoding ribosomal proteins and elonga-tion factors. p70S6K is activated by insulin in muscle [1–3], but not in hepatocytes, according to our recent work [4]. In Correspondence to L. Hue, HORM Unit, ICP-UCL 7529, Brussels, Belgium. Fax: + 32 2764 75 07, Tel.: + 32 2764 75 76, E-mail: hue@horm.ucl.ac.be Abbreviations: ACC, acetyl-CoA carboxylase; AICAr, 5-aminoimi-dazole-4-carboxyamideribonucleoside;ZMP,AICA-ribotide;GAPP, glutamate-activated protein phosphatase; IR, insulin receptor; IRS-1, insulin receptorsubstrate-1; mTOR, mammalian target of rapamycin; p70S6K,p70ribosomalproteinS6kinase;PDK1,3-phosphoinositide-dependent protein kinase; PKB, protein kinase B; PP2A, protein phosphatase 2A. *Present address: GlaxoSmithKline Biologicals, Research and Development, Rue de l’Institut 89, 1330 Rixensart, Belgium. (Received 19 February 2002, revised 19 June 2002, accepted 25 June 2002) activation of both ACC and p70S6K was blocked or reversed when AMPK was activated. This AMPK acti-vation increased Ser79 phosphorylation in ACC but decreased Thr389 phosphorylation in p70S6K. Protein phosphatase inhibitors prevented p70S6K activation when added prior to the incubation with amino acids, whereas they enhanced p70S6K activation when added after the preincubation with amino acids. It is concluded that (a) AMPK blocks amino-acid-induced activation of ACC and p70S6K, directly by phosphorylating Ser79 in ACC, and indirectly by inhibiting p70S6K phosphorylation, and (b)both activation and inhibition of protein phosphatases are involved in the activation of p70S6K by amino acids. p70S6K adds to an increasing list of targets of AMPK in agreement with the inhibition of energy-consuming bio-synthetic pathways. Keywords: ACC; amino acid; AMPK; p70S6K; protein phosphatase. these cells, p70S6K is activated by amino acids like glutamine and leucine, which act synergistically [4]. How-ever, a crosstalk between insulin and amino acids can be demonstrated with leucine, which enhances insulin signal-ling towards p70S6K in many cell types, including hepato-cytes [3–6]. The mechanism of activation of p70S6K involves a complex sequence of multiple serine/threonine phosphory-lations catalysed by several protein kinases. One of these is the mammalian target of rapamycin (mTOR), which phosphorylates p70S6K on Thr389 and is inhibited by the immunosuppressant rapamycin [7]. Phosphorylation of this site correlates with kinase activity [8]. mTOR may also phosphorylateandthereby inactivatea proteinphosphatase that in turn inactivates p70S6K. Indeed, several studies suggest that the amino-acid signalling pathway leading to p70S6K activation comprises inhibition of a protein phos-phatase [9,10]. Whatever the mechanism of activation of p70S6KbymTOR,thelatterplaysanessentialrole,because the activation of p70S6K caused by almost all stimuli so far tested is inhibited by rapamycin. Phosphorylation of Ser411, Thr421 and Ser424, which are within a Ser-Pro rich region located in the autoinhibitory domain, is also thought to modulate p70S6K activity [8,11]. In response to insulin, the 3-phosphoinositide-dependent protein kinase 3752 U. Krause et al. (Eur. J. Biochem. 269) (PDK1)is directly involved in p70S6K activation [11]. The target phosphorylation site for PDK1 is Thr229 in the catalytic domain of p70S6K. A role for protein kinase B (PKB)in the insulin-stimulated activation process of p70S6K has also been proposed [12], but has been ruled out for the amino-acid-induced activation of p70S6K in liver cells [4]. Acetyl-CoAcarboxylase(ACC)isa regulatoryenzymein fatty acid synthesis (reviewed in [13–15]). We have shown that in liver cells ACC activation is correlated with cell swellingbeitinducedbyamino acidsthatarecotransported with Na+ or by hypotonic medium [16]. The activity of ACC is controlled by various mechanisms, including changes in the degree of polymerization, allosteric regula-tion by citrate and glutamate, and covalent modification by phosphorylation/dephosphorylation [13–18]. It is generally assumed that the active form is dephosphorylated, although phosphorylation has been invoked to explain ACC activa-tion by insulin in adipocytes [19]. Under stress conditions, such as anoxia or inhibition of mitochondrial oxidative phosphorylation, the ATP balance becomes negative and, as a result, the AMP/ATP ratio increases. This leads to the activation of the AMP-activated protein kinase (AMPK), which functions as a metabolic master switch and inhibits anabolic processes, thereby preserving ATP (reviewed in [20–22]). ACC is phosphory-lated in vitro by AMPK on Ser79, Ser1200 and Ser1250, the phosphorylation of Ser79 being responsible for inacti-vation [23]. AMPK-inactivated ACC can be reactivated by a glutamate-dependent type-2A protein phosphatase (GAPP), which dephosphorylates a synthetic peptide encompassing the Ser79 phosphorylation site for AMPK in ACC[24].Itis expectedthatinhepatocytes theactivation state of ACC results from the balance between the activities of GAPP and AMPK, although the involvement of other protein kinase or phosphatases has not been ruled out. Because ACC and p70S6K display a similar and parallel pattern of activation in hepatocytes incubated with gluta-mine [4], the question arises whether there is also a common mechanism for inactivation. It is indeed expected that ACC and p70S6K, which control energy-consuming biosynthetic pathways, are less active when ATP supply becomes limiting. Therefore, the effect of different activators of AMPK and the effect of inhibitors of protein phosphatases on the amino-acid-induced activation of ACC and p70S6K were examined in freshly prepared rat hepatocytes. Our results show that the activation of ACC and p70S6K depend on a protein phosphatase and that both enzymes may be inactivated under conditions leading to AMPK activation. MATERIALS AND METHODS Materials 5-Aminoimidazole-4-carboxyamideribonucleoside(AICAr) and oligomycin were from Sigma. Okadaic acid and calyculin A were from Calbiochem. The peptides corres-ponding to the p70S6K substrate [4] and the AMPK substrate (SAMS)[25] were kindly provided by V. Stroo-bant (Ludwig Institute, Brussels, Belgium). Antibodies raised against a peptide containing phosphoSer79 of ACC was a generous gift from D. G. Hardie (Dundee, Scotland). Ó FEBS 2002 Antibodies raised against synthetic phosphopeptides corres-ponding to p70S6K phosphorylation sites containing phos-phoThr389 (anti-pThr389), phosphoSer-411 (anti-pSer411), or phosphoThr421 together with phosphoSer424 [anti-(ppThr421 + Ser424)] were purchased from Santa Cruz. The source of all other materials is given in [4,26]. Hepatocytes preparation and incubation Hepatocytes from overnight-fasted male Wistar rats (170– 200 gofbodyweight)werepreparedasdescribedpreviously [4] and incubated at 37 °C for the indicated periods of time following15 minpreincubation,ataconcentrationofabout 50 mgÆmL)1 in Krebs–Henseleit bicarbonate buffer in equilibrium with a 95% O2/5% CO2 gas phase in the presence of 20 mM glucose and other substances as indicatedinthelegendstotheFigures.Anoxiawasobtained by incubating the cells in a 95% N2/5% CO2 gas phase. At the end of the incubations, the cells were collected by centrifugation (2 s, microfuge)and the cell pellets were immediately stored in liquid nitrogen. The cell pellets were homogenized in 0.5 mL of the lysis buffer as described previously [4]. After centrifugation (20 000 g, 15 min) , the supernatants were stored at )80 °C. Enzyme assays Methods for the measurements of the activity of AMPK after precipitation with 6% (w/v)polyethylene glycol 6000, of ACC and p70S6K, and for immunoprecipitation of p70S6K from cell extracts have been described [4,27,28]. ACC was measured in the presence of 0.5 mM citrate-Mg [27]. One unit of enzyme activity corresponds to 1 nmol (protein kinases)or 1 lmol (ACC)of product formed per min under the assay conditions. Other methods The phosphorylation state of p70S6K in hepatocytes was evaluated by gel mobility shift assay [4] as well as by immunoblots with antiphosphopeptides. In vitro trials of p70S6K phosphorylation by purified AMPK were per-formed as follows. Immunoprecipitates of p70S6K from extractsofcontrolandamino-acid-treatedhepatocyteswere incubated at 30 °C for 30 min in a total volume of 50 lL in the presence of 60 mU of purified liver AMPK [26] with or without 2 mM AMP and 100 lM [c-32P]ATP-Mg (3000 c.p.m.Æpmol)1)for the measurement of the incorpo-ration of radioactive phosphate after SDS/PAGE and autoradiography, or 1 mM ATP-Mg for the measurement of p70S6K activity [4]. RESULTS AMPK is involved in the inactivation of ACC and p70S6K In agreement with our previous studies [4], there was a similar pattern of activation of ACC and p70S6K in hepatocytes incubated with glutamine, suggesting a com-mon point of control in their signalling pathways (Fig. 1). The effects of AMPK activation on p70S6K and ACC were compared. AMPK was activated by incubating the cells under stress conditions, namely anoxia (Fig. 1), AICAr or Ó FEBS 2002 Inhibition of p70S6K activation by AMPK (Eur. J. Biochem. 269)3753 Fig. 2. Effect of AICAr and oligomycin on ACC, p70S6Kand AMPK activities. The experimental protocol is shown schematically at the top of the figure. After an equilibrium period of 15 min, hepatocytes were incubated under control conditions or in the presence of 10 mM glu-tamine for 50 min (open bars). The cells were then further incubated with 0.5 mM AICAr or 1 lM oligomycin for 10 min (filled bars). Maximal activation was seen for ACC and p70S6K after a 60-min incubation with glutamine (32.8 ± 2.8 and 348 ± 34 mUÆg)1 of cells, respectively; means ± SEM, n ¼ 3)and for AMPK after a 10-min incubation with AICAr or oligomycin (24.5 ± 2.1 and 22.8 ± 1.3 UÆg)1 of cells, respectively; means ± SEM, n ¼ 3). The values are expressed as percent of maximal activity observed for each enzyme. Gln, glutamine; Oligo, oligomycin. Fig. 1. Time-course of the effect of anoxia on ACC, p70S6Kand AMPKactivities. Hepatocyteswereincubatedfortheindicatedperiods of time under control conditions (s, Ctr), in the presence of 10 mM glutamine (n, Gln), under a nitrogen atmosphere (,, N2)or in the presence of 10 mM glutamine under a nitrogen atmosphere (d, Gln + N2). The values are the means ± SEM for three cell prepa- rations. oligomycin, an inhibitor of mitochondrial oxidative phos-phorylation (Fig. 2). Anoxia and oligomycin increase intracellular AMP concentration, whereas AICAr is phos-phorylated into AICA-ribotide (ZMP), an AMP analogue. Both ZMP and AMP are activators of AMPK. As already observed in heart [26], AMPK activation by anoxia was transient,beingmaximalat10 minbeforereturningtobasal or even lower values between 45 and 60 min of incubation (Fig. 1).Likeanoxia,bothAICArandoligomycinactivated AMPK (Fig. 2)and this activation was not changed in hepatocytesincubatedwithglutamine(Figs 1and2).Under thesestressconditions,AMPKactivationledtoinactivation of both ACC and p70S6K, suggesting that AMPK activa-tion overruled the control by amino acids. However, when AMPK activity returned towards basal levels at 45 and 60 min, ACC but not p70S6K started to reactivate. These 3754 U. Krause et al. (Eur. J. Biochem. 269) Ó FEBS 2002 data suggest that AMPK plays a role in the inactivation process of ACC and p70S6K under metabolic stress conditions. Rapamycin and AICAr exert different effects on the activation of ACC and p70S6K induced by glutamine RapamycinisapotentinhibitorofmTORandoftheinsulin-dependent activation of p70S6K in skeletal muscle [29–31]. We compared the time-course of the effect of AICAr with that of rapamycin on ACC and p70S6K, both of which had been activated by a 50-min incubation with glutamine (Fig. 3). Rapamycin inactivated p70S6K but was without effectonACC,inagreementwithourpreviousfindings[27]. ThisrulesoutaroleformTORorp70S6Kintheglutamine-mediatedactivationofACC.Theinactivationofp70S6Kby rapamycin occurred within seconds ()27% after 20 s)and was complete between 5 and 10 min of incubation. In contrast, the inactivation of p70S6K by AICAr was slower and was half-maximal only at 7 min, whereas the inactiva-tion of ACC by AICAr was half-maximal at about 1 min (Fig. 3). The velocity of the onset of ACC inactivation indicatesthatAICArisquicklytransportedintothehepato-cytesandindeedleadstoanimmediateactivationofAMPK (Fig. 3), which in turn inactivates ACC by phosphorylating Ser79 (Fig. 4). The comparison of the sensitivity of ACC, p70S6K and AMPK towards AICAr showed that half-maximaleffectswereobservedatabout30 lM forACCand 110 lM for AMPK and p70S6K (Fig. 4). ACC activity and Ser79 phosphorylation Phosphorylation of Ser79 is known to inactivate ACC by decreasing the Vmax [18,23]. In vitro, this site is phospho-rylated by AMPK and dephosphorylated by GAPP. Immunoblotting hepatocyte extracts with an anti-phospho-peptide (anti-phosphoSer79 Ig)demonstrated that Ser79 was indeed phosphorylated (Fig. 4)when AMPK was activated. We confirmed that this ACC inactivation corre-spondedtoadecreaseinVmax (datanotshown).Incontrast, ACC activation by amino acids occurred without a change in Ser79 phosphorylation (Fig. 4), although it was blocked by protein phosphatase inhibitors (see below). This suggests thatdephosphorylationoccursatothersitesonACCorthat the process is indirect. P70S6K is not a direct substrate of AMPK We tested the possibility of a direct phosphorylation and inactivation of p70S6K by AMPK in vitro. p70S6K purified from control hepatocytes or from cells treated with amino acidscouldnotbe phosphorylated bypurifiedAMPK (data not shown). In addition, attempts to inactivate p70S6K by AMPK in vitro also failed (without/with AMPK: control, 14/14; glutamine, 129/111; glutamine and leucine, 530/ 500 mUÆg cells)1, n ¼ 2). Inhibitors of protein phosphatases exert different effects on ACC and p70S6K activities Theeffectoftwoinhibitorsofproteinphosphatases,namely okadaic acid and calyculin A, were investigated. Both inhibitors are cell permeable compounds with tumor Fig. 3. Time-course of the effects of rapamycin or AICAr on ACC, p70S6Kand AMPKactivities. The experimental protocol is shown schematically at the top of the figure. Hepatocytes were incubated in the presence of 10 mM glutamine (Gln)to activate ACC and p70S6K. After 50 min, NaCl (0.9% final concentration, n), rapamycin (Rapa, 300 nM final concentration, .)or AICAr (0.5 m M final concentration, j)were added and the cells were further incubated for up to 30 min. The values are the means ± SEM for three cell preparations. Ó FEBS 2002 Inhibition of p70S6K activation by AMPK (Eur. J. Biochem. 269)3755 Fig. 4. Dose–response curve of the effect of AICAr on ACC, p70S6Kand AMPKactivities and Ser79 phosphorylation in ACC. The experimental protocol is shown schematically at the bottom right of the figure. Hepatocytes were incubated under control conditions or stimulated with 10 mM glutamine.After50 min,AICArwasaddedandthecellswerefurtherincubatedfor10 min(samplingA:ACCandAMPK)or20 min(samplingB: p70S6K). AICAr (h, Ctr); glutamine plus AICAr (j, Gln). The values of enzyme activity are the means ± SEM for three cell preparations. Ser79 phosphorylation was detected by immunoblotting extracts (15 lg of proteins)with the anti-phosphoSer79 Ig (lower left panel)and was quantified by densitometry (middle left panel, mean of two experiments). promoting properties that target on the serine/threonine protein phosphatases PP2A and PP1 [32]. Preincubation of hepatocytes with okadaic acid prevented the activation of ACC (100%)and of p70S6K (by 70%)in hepatocytes incubated with glutamineplus leucine(Fig. 5). These results suggest that a protein phosphatase is required for the activation of both ACC and p70S6K by amino acids. The effectoftheseinhibitorsdifferedwhentheywereaddedafter a preincubation with amino acids to activate both enzymes. Under these conditions, okadaic acid inactivated ACC, whereas it enhanced p70S6K activation (Fig. 5). Similar results were obtained with calyculin A (data not shown). The phosphorylation state of p70S6K The activation of p70S6K involves multiple phosphoryla-tions of the protein [1–3]. The phosphorylated forms can be detected by their reduced mobility during SDS/PAGE and by blotting with anti-phosphopeptides (Fig. 6). The pro-portion of slow electrophoretic, phosphorylated forms of p70S6K that appeared after stimulation of the cells with glutamine or glutamine plus leucine correlated with the increase in p70S6K activity brought about by these amino acids (Fig. 6, lanes 1–4). The data also show that increases in p70S6K activity correlate with increases in Thr389, Thr421 and Ser424 phosphorylation state, whereas Ser411 phosphorylation was unaffected (Fig. 6). AICAr did not change the phosphorylation state of p70S6K if compared with the controls (Fig. 6, compare lane 5 with lane 1), but it counteracted phosphorylation induced by amino acids and drastically decreased Thr389 phosphorylation (Fig. 6, com-pare lane 7 with lane 4). This suggests that AMPK did not directly target on p70S6K, thus confirming our lack of experimental evidence in vitro for a direct phosphorylation of p70S6K by AMPK. Addition of calyculin A after a preincubation with amino acids to activate p70S6K resulted in a further activation of p70S6K (Figs 5 and 6), which corresponded to the appearance of the slowest electropho-retic forms and maximal phosphorylation of Thr389, Thr421 and Ser424. (Fig. 6, compare lane 8 with lane 4). ... - tailieumienphi.vn